Thrombosis, complicating a ruptured plaque, is ultimately responsible for the majority of cardiac mortality and morbidity in the Western World, including most instances of heart failure. The hypothesis driving this" work is that gene transfer of tissue factor pathway inhibitor (TFPI) to mechanically damaged atherosclerotic arteries and to vein grafts will suppress the development of chronic vascular stenosis and vein graft deterioration by interrupting tissue factor and thrombin-dependent mechanisms of recurrent thrombosis and intimal hyperplasia. In addition, we hypothesize that treatment of injured arteries and grafted veins with both the TFPI and cyclooxygenase-1 (PGHS-1) genes will further improve the protection of the vascular conduits" against thrombosis, vasospasm, and vascular smooth muscle cell accumulation. Tissue factor (TF) is the cellular initiator of thrombin generation, which drives fibrinogen clotting and platelet activation, resulting in the release of mitogens to the injured vessel wall. TF promotes directly vascular smooth muscle cell (VSMC) migration, whereas factor Xa and thrombin are VSMC mitogens. Tissue factor pathway inhibitor (TFPI), an endogenous inhibitor of TF and factor Xa, can inhibit thrombosis and neointima formation after vascular injury but prolonged therapy at the high doses needed entails bleeding risks. Here we propose to evaluate TFPI gene transfer as a means to silence TF and to reduce lesion formation in balloon-injured atherosclerotic rabbit arteries and grafted rabbit jugular veins. In preliminary work, we describe how adenoviral gene transfer of TFPI reduced thrombosis and intimal hyperplasia in a porcine and Watanabe rabbit carotid injury model. We show that combined gene transfer of TFPI and PGHS-1 further improves blood flow in these models by suppressing thrombosis and promoting vasodilation. Given the problems with adenoviral vectors, we propose the use of adeno-associated virus (rAAV)-mediated gene transfer, associated with prolonged transgene expression and little inflammatory risk. We will assess the outcome of TFPI and PGHS-1 gene transfer with Doppler evaluation of blood flow, quantitative histomorphometry, and in situ assessment of prothrombin/fibrin deposition and prostacyclin/cAMP synthesis and will examine whether local overexpression of TFPI attenuates the thrombogenicity of the injured atherosclerotic vessel wall itself. Our studies will further elucidate the role of TF in restenosis and vein graft disease in atherosclerotic states and provide new strategies for these vascular disorders, which are a major health and cost problem in the Western World. We hypothesize that gene transfer of TFPI and PGHS-1 will achieve lasting vasoprotection without hemorrhagic risk.